Enzyme sensors are sensors where a chemical species to be measured (an analyte) undergoes an enzyme catalysed reaction in the sensor before detection. The reaction between the analyte and the enzyme (for which the analyte should be a substrate) yields a secondary species which concentration (under ideal conditions) is proportional with or identical to the concentration of the analyte. The concentration of the secondary species is then detected by a transducer, e.g., by means of an electrode.
The enzyme of an enzyme sensor is typically included in a membrane suited for contacting the test fluid. The enzyme may be included as part of a membrane of a sensor or may be located behind such a membrane. Another alternative is to incorporate the enzyme into the sensor proper, e.g. as part of a carbon paste layer of an electrode. Hence, the analyte is contacted with the enzyme after diffusion into the outer part of the sensor (e.g. the membrane or the carbon paste layer), the enzyme/analyte reaction takes place, and the secondary species then diffuses to the detector part of the sensor, e.g. an electrode.
In a traditional enzyme sensor comprising a membrane, the membrane must, on the one hand, have a suitable porosity so that the analyte diffuses from the test fluid to the enzyme in a controlled manner, and, on the other hand, be impermeable or substantially impermeable to the enzyme in question in order to avoid leaching of the enzyme into the test fluid.
One way to overcome this problem is to immobilize the enzyme in question to a macromolecule so that the physical size and the general low solubility of the enzyme/macromolecule prohibit leaching through the porous membrane. A possible alternative, which may be more suited for in-situ polymerized membranes, could be to entrap the enzyme in polymers.
An interesting example of an analyte which is present in, e.g., body fluids is lactate. Lactate sensors wherein lactate oxidase is immobilized to bovine serum albumin are known (Hu et al., 1993, Analytica Chimica Acta, 281, pp 503-511; Tsuchida et al., 1985, Biotechnology and Bioengineering, 27, pp 837-841; Pfeiffer et al., 1992, Biosensors & Bioelectronics, 7, pp 661-671; Baker et al., 1995, Anal. Chem., 67, pp 1536-1540; and Liu et al., 1995, Electrochemica Acta, 40, pp 1845-1849). The reported lifetime of lactate sensors is typically around 14 days (see for example Winckers et al., 1996, Clinical Chemistry, 42, No. S6, p S278, poster No. 761).
Typically, when an enzyme, e.g. lactate oxidase, is immobilized, the initial specific enzymatic activity of the immobilized enzyme is reduced compared to a corresponding amount of non-immobilized enzyme. This may be due to the fact that the enzyme, or at least a part (a domain) thereof, does not retain its active conformation during or after the immobilization reaction, or that the immobilization reaction involves functional groups of the enzyme which are situated in or very close to the active site of the enzyme. Thus, a considerable amount of enzyme may be inactivated in the immobilization reaction. The presence of large amounts of inactivated, partially inactivated or denatured enzyme in a sensor is highly undesirable as this may lead to longer response times.
It has been reported that immobilization of trypsin to a solid phase material in the presence of a competitive inhibitor results in almost full retention of the initial specific enzymatic activity (Kuga et al., 1976, Mem. Fac. Sci. Kyushu Univ. Ser. C, 10, pp 77-90; CA: 85:42969h). It has also been described to use a polymeric competitive inhibitor when immobilizing trypsin to water-insoluble carriers (Brown et al., 1981, Makromol. Chem., 182, pp 1605-1616). Neither of these publications addresses the use of immobilized trypsin in an enzyme sensor, and, to applicant's best knowledge, no other publications address the use of immobilized trypsin in an enzyme sensor. Thus, the special conditions and requirements applying for immobilized enzymes for enzyme sensors have not been addressed or rendered obvious.
The aim of the present invention is to provide enzyme sensors which have an improved initial specific enzymatic activity. Another aim of the present invention is to provide a method for stabilizing the enzymatic activity of enzyme sensors so that the lifetime of enzyme sensors can be prolonged.